Several publications and patent documents are cited throughout the specification in order to more fully describe the state of the art to which this invention pertains. The disclosure of each of these publications and patent documents is incorporated by reference herein.
Rapamycin (sirolimus) (FIG. 1) is a lipophilic macrolide produced by Streptomyces hygroscopicus NRRL 5491 (Sehgal et al., 1975; Vézina et al., 1975; U.S. Pat. Nos. 3,929,992; 3,993,749) with a 1,2,3-tricarbonyl moiety linked to a pipecolic acid lactone (Paiva et al., 1991). For the purpose of this invention rapamycin is described by the numbering convention of McAlpine et al. (1991) in preference to the numbering conventions of Findlay et al. (1980) or Chemical Abstracts (11th Cumulative Index, 1982-1986 p60719CS).
Rapamycin has significant therapeutic value due to its wide spectrum of biological activities (Huang et al., 2003). The compound is a potent inhibitor of the mammalian target of rapamycin (mTOR), a serine-threonine kinase downstream of the phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B) signalling pathway that mediates cell survival and proliferation. This inhibitory activity is gained after rapamycin binds to the immunophilin FK506 binding protein 12 (FKBP12) (Dumont, F. J. and Q. X. Su, 1995). In T cells rapamycin inhibits signalling from the IL-2 receptor and subsequent autoproliferation of the T cells resulting in immunosuppression. Rapamycin is marketed as an immunosuppressant for the treatment of organ transplant patients to prevent graft rejection (Huang et al, 2003). In addition to immunosuppression, rapamycin has potential therapeutic use in the treatment of a number of diseases, for example, cancer, cardiovascular diseases such as restenosis, autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, fungal infection and neurodegenerative diseases such as Parkinson's disease and Huntington's diseases.
Despite its utility in a variety of disease states rapamycin has a number of major drawbacks. Firstly it is a substrate of cell membrane efflux pump P-glycoprotein (P-gp, LaPlante et al, 2002, Crowe et al, 1999) which pumps the compound out of the cell making the penetration of cell membranes by rapamycin poor. This causes poor absorption of the compound after dosing. In addition, since a major mechanism of multi-drug resistance of cancer cells is via cell membrane efflux pump, rapamycin is less effective against multi-drug resistance (MDR) cancer cells. Secondly rapamycin is extensively metabolised by cytochrome P450 enzymes (Lampen et al, 1998). Its loss at hepatic first pass is high, which contributes further to its low oral bioavailability. The role of CYP3A4 and P-gp in the low bioavailability of rapamycin has been confirmed in studies demonstrating that administration of CYP3A4 and/or P-gp inhibitors decreased the efflux of rapamycin from CYP3A4-transfected Caco-2 cells (Cummins et al, 2004) and that administration of CYP3A4 inhibitors decreased the small intestinal metabolism of rapamycin (Lampen et al, 1998). The low oral bioavailability of rapamycin causes significant inter-individual variability resulting in inconsistent therapeutic outcome and difficulty in clinical management (Kuhn et al, 2001, Crowe et al, 1999).
Therefore, there is a need for the development of novel rapamycin-like compounds that are not substrates of P-gp, that may be metabolically more stable and therefore may have improved bioavailability. When used as anticancer agents, these compounds may have better efficacy against MDR cancer cells, in particular against P-gp-expressing cancer cells.
A range of synthesised rapamycin analogues using the chemically available sites of the molecule has been reported. The description of the following compounds was adapted to the numbering system of the rapamycin molecule described in FIG. 1. Chemically available sites on the molecule for derivatisation or replacement include C40 and C28 hydroxyl groups (e.g. U.S. Pat. Nos. 5,665,772; 5,362,718), C39 and C16 methoxy groups (e.g. WO 96/41807; U.S. Pat. No. 5,728,710), C32, C26 and C9 keto groups (e.g. U.S. Pat. Nos. 5,378,836; 5,138,051; 5,665,772). Hydrogenation at C17, C19 and/or C21, targeting the triene, resulted in retention of antifungal activity but relative loss of immunosuppression (e.g. U.S. Pat. Nos. 5,391,730; 5,023,262). Significant improvements in the stability of the molecule (e.g. formation of oximes at C32, C40 and/or C28, U.S. Pat. Nos. 5,563,145, 5,446,048), resistance to metabolic attack (e.g. U.S. Pat. No. 5,912,253), bioavailability (e.g. U.S. Pat. Nos. 5,221,670; 5,955,457; WO 98/04279) and the production of prodrugs (e.g. U.S. Pat. Nos. 6,015,815; 5,432,183) have been achieved through derivatisation.
Two of the most advanced rapamycin derivatives in clinical development are 40-O-(2-hydroxy)ethyl-rapamycin (RAD001, Certican, everolimus) a semi-synthetic analogue of rapamycin that shows immunosuppressive pharmacological effects (Sedrani, R. et al., 1998; Kirchner et al., 2000; U.S. Pat. No. 5,665,772) and 40-O[2,2-bis(hydroxymethyl)propionyoxy]rapamycin, CCI-779 (Wyeth-Ayerst) an ester of rapamycin which inhibits cell growth in vitro and inhibits tumour growth in vivo (Yu et al., 2001). CCI-779 is currently in Phase III clinical trials. Studies investigating the pharmacokinetics of RAD001 have shown that, similarly to rapamycin, it is a substrate for P-gp (Crowe et al, 1999, LaPlante et al, 2002).
The present invention provides the novel and surprising use of 39-desmethoxyrapamycin in medicine, particularly in the treatment of cancer or B-cell malignancies, in the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, diseases of inflammation vascular disease and fibrotic diseases. In particular the present invention provides for the use of 39-desmethoxyrapamycin in the treatment of cancer and B-cell malignancies. Despite its close structural similarity to rapamycin the compound of the invention displays a surprisingly different pharmacological profile. In particular it has significantly increased cell membrane permeability and decreased efflux in comparison with rapamycin, and it is not a substrate for P-gp. Additionally, this compound shows more potent activity against multi-drug resistant and P-gp-expressing cancer cell lines than rapamycin. When compared with rapamycin it shows a significantly different inhibitory profile against the NCI 60 cell line panels. 39-Desmethoxyrapamycin also shows increased metabolic stability with respect to rapamycin.
Therefore, the present invention provides for the medical use of 39-desmethoxyrapamycin, a rapamycin analogue with improved metabolic stability, improved cell membrane permeability, a decreased rate of efflux and a different tumour cell inhibitory profile to rapamycin. This compound is useful in medicine, in particular for the treatment of cancer and/or B-cell malignancies, in the induction or maintenance of immunosuppression, the stimulation of neuronal regeneration or the treatment of fungal infections, transplantation rejection, graft vs. host disease, autoimmune disorders, diseases of inflammation vascular disease and fibrotic diseases. The present invention particularly provides for the use of 39-desmethoxyrapamycin in the treatment of cancer and/or B-cell malignancies.